The present invention relates generally to plant molecular biology. In particular, it relates to nucleic acids and methods for conferring pest resistance in plants, particularly lettuce.
Recently, several resistance genes have been cloned by several groups from several plants. Many of these genes are sequence related. The derived amino acid sequences of the most common class, RPS2, RPM1 (bacterial resistances in Arabidopsis (Mindrinos et al. Cell 78:1089-1099 (1994)); Bent et al. Science 265:1856-1860 (1994); Grant et al., Science 269:843-846 (1995)), L6 (fungal resistance in flax; Lawrence, et al., The Plant Cell 7:1195-1206 (1995)), and N, (virus resistance in tobacco; Whitham, et al., Cell 78:1101-1115 (1994); and U.S. Pat. No. 5,571,706), all contain leucine-rich repeats (LRR) and nucleotide binding sites (NBS).
The NBS is a common motif in several mammalian gene families encoding signal transduction components (e.g., Ras) and is associated with ATP/GTP-binding sites.
The NBS is a common motif in several mammalian gene families encoding signal transduction components (e.g., Ras) and is associated with ATP/GTP-binding sites.
LRR domains can mediate protein-protein interactions and are found in a variety of proteins involved in signal transduction, cell adhesion and various other functions. LRRs are leucine rich regions often comprising 20-30 amino acid repeats where leucine and other aliphatic residues occur periodically. LRRs can function extracellularly or intracellularly.
Since the onset of civilization, plant diseases have had catastrophic effects on crops and the well-being of the human population. Plant diseases continue to effect enormous human and economic costs. An increasing human population and decreasing amounts of arable land make all approaches to preventing and treating plant pathogen destruction critical. The ability to control and enhance a plant""s protective responses against pathogens would be of enormous benefit. Tissue-specific and temporal control of mechanisms responsible for plant cell death would also be of great practical and economic value. The present invention fulfills these and other needs.
What is needed in the art are plant disease resistance genes and means to create transgenic disease resistance plants, particularly in lettuce. Further, what is needed in the art is a means to DNA fingerprint cultivars and germplasm with respect to their disease resistance haplotypes for use in plant breeding programs. The present invention provides these and other advantages.
The present invention provides isolated nucleic acid constructs. These constructs comprise an RG (resistance gene) polynucleotide which encodes an RG polypeptide having at least 60% sequence identity to an RG polypeptide selected from the group consisting of: an RG1 polypeptide, an RG2 polypeptide, an RG3 polypeptide, and an RG4 polypeptide. RG1, RG2, RG3, RG4, and the like, represent individual xe2x80x9cRG families.xe2x80x9d Each xe2x80x9cRG family,xe2x80x9d as defined herein, is a group of polypeptide sequences that have at least 60% amino acid sequence identity. Individual members of an RG family, i.e., individual species of the genus, typically map to the same genomic locus. The invention provides for constructs comprising nucleotides encoding the RG families of the invention, which can include sequences encoding a leucine rich region (LRR), and/or a nucleotide binding site (NBS), or both.
The invention provides for an isolated nucleic acid construct comprising an RG polynucleotide which encodes an RG polypeptide having at least 60% sequence identity to an RG polypeptide from an RG family selected from the group consisting of: an RG1 polypeptide, an RG2 polypeptide, an RG3 polypeptide, an RG4 polypeptide, an RG5 polypeptide, and an RG7 polypeptide. In alternative embodiments, the nucleic acid construct comprises an RG polynucleotide which encodes an RG polypeptide comprising an leucine rich region (LRR), or, an RG polypeptide comprising a nucleotide binding site (NBS). The nucleic acid construct can comprise a polynucleotide which is a full length gene. In another embodiment, the nucleic acid construct encodes a fusion protein.
In one embodiment, the nucleic acid construct comprises a sequence encoding an RG1 polypeptide. The RG1 polypeptide can be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO:1 (RG1A), SEQ ID NO:2 and SEQ ID NO:137 (RG1B), SEQ ID NO:3 (RG1C), SEQ ID NO:4 (RG1D), SEQ ID NO:5 (RG1E), SEQ ID NO:6 (RG1F), SEQ ID NO:7 (RG1G), SEQ ID NO:8 (RG1H), SEQ ID NO:9 (RG1I), and SEQ ID NO:10 (RG1J).
In another embodiment, the nucleic acid construct comprises a sequence encoding an RG2 polypeptide. The RG2 polypeptide can be encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO:21 and SEQ ID NO:27 (RG2A); SEQ ID NO:23 and SEQ ID NO:28 (RG2B); SEQ ID NO:29 (RG2C); SEQ ID NO:30 (RG2D); SEQ ID NO:31 (RG2E); SEQ ID NO:32 (RG2F); SEQ ID NO:33 (RG2G); SEQ ID NO:34 (RG2H); SEQ ID NO:35 (RG21); SEQ ID NO:36 (RG2J); SEQ ID NO:37 (RG2K); SEQ ID NO:38 (RG2L); SEQ ID NO:39 (RG2M); SEQ ID NO:87 (RG2A); SEQ ID NO:89 (RG2B); SEQ ID NO:91 (RG2C); SEQ ID NO:93 (RG2D) and SEQ ID NO:94 (RG2D); SEQ ID NO:96 (RG2E); SEQ ID NO:98 (RG2F); SEQ ID NO:100 (RG2G); SEQ ID NO:102 (RG2H); SEQ ID NO:104 (RG21); SEQ ID NO:106 (RG2J) and SEQ ID NO:107 (RG2J); SEQ ID NO:109 and SEQ ID NO:110(RG2K); SEQ ID NO:112 (RG2L); SEQ ID NO:114 (RG2M); SEQ ID NO:116 (RG2N); SEQ ID NO:118 (RG2O); SEQ ID NO:120 (RG2P); SEQ ID NO:122 (RG2Q); SEQ ID NO:124 (RG2S); SEQ ID NO:126 (RG2T); SEQ ID NO:128 (RG2U); SEQ ID NO:130 (RG2V); and, SEQ ID NO:132 (RG2W).
In other embodiments, the nucleic acid construct comprises a RG3 sequence (SEQ ID NO:68) encoding an RG3 polypeptide (SEQ ID NO:138) (RG3). In other embodiments, the nucleic acid construct comprises an RG4 sequence (SEQ ID NO:69) encoding an RG4 polypeptide (SEQ ID NO:139) (RG4).
In other embodiments, the nucleic acid construct comprises a RG5 sequence (SEQ ID NO:134) encoding an RG5 polypeptide (SEQ ID NO:135). The RG5 polypeptide can be encoded by a polynucleotide sequence as set forth in SEQ ID NO:134.
The invention also provides for a nucleic acid construct which comprises an RG7 sequence encoding an RG7 polypeptide. The RG7 polypeptide can be encoded by a polynucleotide sequence as set forth in SEQ ID NO:136.
In further embodiments, the nucleic acid construct can further comprise a promoter operably linked to the RG polynucleotide. In alternative embodiments, the promoter can be a plant promoter; a disease resistance promoter; a lettuce promoter; a constitutive promoter; an inducible promoter; or, a tissue-specific promoter. The nucleic acid construct can comprise a promoter sequence from an RG gene linked to a heterologous polynucleotide.
The invention also provides for a transgenic plant comprising a recombinant expression cassette comprising a promoter operably linked to an RG polynucleotide. The expression cassette can comprise a plant promoter or a viral promoter; the plant promoter can be a heterologous promoter. In one embodiment, the transgenic plant is lettuce. In alternative embodiments, the transgenic plant comprises an expression cassette which includes an RG polynucleotide selected from the group consisting of SEQ ID NO:1 (RG1A); SEQ ID NO:2 and SEQ ID NO:137 (RG1B); SEQ ID NO: 3 (RG1C); SEQ ID NO:4 (RG1D); SEQ ID NO:5 (RG1E); SEQ ID NO:6 (RG1F); SEQ ID NO:7 (RG1G); SEQ ID NO:8 (RG1H); SEQ ID NO:9 (RG1I) and SEQ ID NO:10 (RG1J); SEQ ID NO:21 and SEQ ID NO:27 (RG2A); SEQ ID NO:23 and SEQ ID NO:28 (RG2B); SEQ ID NO:29 (RG2C); SEQ ID NO:30 (RG2D); SEQ ID NO:31 (RG2E); SEQ ID NO:32 (RG2F); SEQ ID NO:33 (RG2G); SEQ ID NO:34 (RG2H); SEQ ID NO:35 (RG2I); SEQ ID NO:36 (RG2J); SEQ ID NO:37 (RG2K); SEQ ID NO:38 (RG2L); SEQ ID NO:39 (RG2M); SEQ ID NO:87 (RG2A); SEQ ID NO:89 (RG2B); SEQ ID NO:91 (RG2C); SEQ ID NO:93 (RG2D) and SEQ ID NO:94 (RG2D); SEQ ID NO:96 (RG2E); SEQ ID NO:98 (RG2F); SEQ ID NO: 100 (RG2G); SEQ ID NO:102 (RG2H); SEQ ID NO:104 (RG2I); SEQ ID NO:106 (RG2J) and SEQ ID NO: 107 (RG2J); SEQ ID NO:109 and SEQ ID NO:110(RG2K); SEQ ID NO:112 (RG2L); SEQ ID NO:114 (RG2M); SEQ ID NO:116 (RG2N); SEQ ID NO:118 (RG20); SEQ ID NO:120 (RG2P); SEQ ID NO:122 (RG2Q); SEQ ID NO:124 (RG2S); SEQ ID NO:126 (RG2T); SEQ ID NO:128 (RG2U); SEQ ID NO:130 (RG2V); and, SEQ ID NO:132 (RG2W); SEQ ID NO:68 (RG3); SEQ ID NO:69 (RG4); SEQ ID NO:134 (RG5); or SEQ ID NO:136 (RG7).
The invention provide for a transgenic plant comprising an expression cassette comprising an RG polynucleotide which can encode an RG1 polypeptide selected from the group consisting of SEQ ID NO:11 (RG1A), SEQ ID NO:12 (RG1B), SEQ ID NO:13 (RG1C), SEQ ID NO:14 (RG1D), SEQ ID NO:15 (RG1E), SEQ ID NO:16 (RG1F), SEQ ID NO:17 (RG1G), SEQ ID NO:18 (RG1H), SEQ ID NO:19 (RG1I), or SEQ ID NO:20 (RG1J); or, an RG2 polypeptide selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:41 (RG2A); SEQ ID NO:24 and SEQ ID NO:42 (RG2B); SEQ ID NO:43 (RG2C); SEQ ID NO:44 (RG2D); SEQ ID NO:45 (RG2E); SEQ ID NO:46 (RG2F); SEQ ID NO:47 (RG2G); SEQ ID NO:48 (RG2H); SEQ ID NO:49 (RG2I); SEQ ID NO:50 (RG2J); SEQ ID NO:51 (RG2K); SEQ ID NO:52 (RG2L); SEQ ID NO:53 (RG2M); SEQ ID NO:88 (RG2A); SEQ ID NO:90 (RG2B); SEQ ID NO:92 (RG2C); SEQ ID NO:95 (RG2D); SEQ ID NO:97 (RG2E); SEQ ID NO:99 (RG2F); SEQ ID NO:101 (RG2G); SEQ ID NO:103 (RG2H); SEQ ID NO:105 (RG2I); SEQ ID NO:108 (RG2J); SEQ ID NO:111 (RG2K); SEQ ID NO:113 (RG2L); SEQ ID NO:115 (RG2M); SEQ ID NO:117 (RG2N); SEQ ID NO:119 (RG20); SEQ ID NO:121 (RG2P); SEQ ID NO:123 (RG2Q); SEQ ID NO:125 (RG2S); SEQ ID NO:127 (RG2T); SEQ ID NO:129 (RG2U); SEQ ID NO:131 (RG2V); and, SEQ ID NO:133 (RG2W); an RG4 polypeptide as set forth by SEQ ID NO:72; an RG5 polypeptide with a sequence as set forth by SEQ ID NO:135; or, an RG7 polypeptide.
The invention also provides for a method of enhancing disease resistance in a plant, the method comprising introducing into the plant a recombinant expression cassette comprising a promoter functional in the plant and operably linked to an RG polynucleotide sequence. In this method, the plant can be a lettuce plant; and, the RG polynucleotide can encode an RG polypeptide selected from the group consisting of an RG1 polypeptide selected from the group consisting of SEQ ID NO:11 (RG1A), SEQ ID NO:12 (RG1B), SEQ ID NO:13 (RG1C), SEQ ID NO:14 (RG1D), SEQ ID NO:15 (RG1E), SEQ ID NO:16 (RG1F), SEQ ID NO:17 (RG1G), SEQ ID NO:18 (RG1H), SEQ ID NO:19 (RG1I), or SEQ ID NO:20 (RG1J); or, an RG2 polypeptide selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:41 (RG2A); SEQ ID NO:24 and SEQ ID NO:42 (RG2B); SEQ ID NO:43 (RG2C); SEQ ID NO:44 (RG2D); SEQ ID NO:45 (RG2E); SEQ ID NO:46 (RG2F); SEQ ID NO:47 (RG2G); SEQ ID NO:48 (RG2H); SEQ ID NO:49 (RG2I); SEQ ID NO:50 (RG2J); SEQ ID NO:51 (RG2K); SEQ ID NO:52 (RG2L); SEQ ID NO:53 (RG2M); SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:88 (RG2A); SEQ ID NO:90 (RG2B); SEQ ID NO:92 (RG2C); SEQ ID NO:95 (RG2D); SEQ ID NO:97 ( RG2E); SEQ ID NO:99 (RG2F); SEQ ID NO:101 (RG2G); SEQ ID NO:103 (RG2H); SEQ ID NO:105 (RG2I); SEQ ID NO:108 (RG2J); SEQ ID NO:111 (RG2K); SEQ ID NO:113 (RG2L); SEQ ID NO:115 (RG2M); SEQ ID NO:117 (RG2N); SEQ ID NO:119 (RG20); SEQ ID NO:121 (RG2P); SEQ ID NO:123 (RG2Q); SEQ ID NO:125 (RG2S); SEQ ID NO:127 (RG2T); SEQ ID NO:129 (RG2U); SEQ ID NO:131 (RG2V); and, SEQ ID NO:133 (RG2W). In this method, the promoter can be a plant disease resistance promoter, a tissue-specific promoter, a constitutive promoter, or an inducible promoter.
The invention also provides for a method of detecting RG resistance genes in a nucleic acid sample, the method comprising: contacting the nucleic acid sample with an RG polynucleotide to form a hybridization complex; and, wherein the formation of the hybridization complex is used to detect the RG resistance gene in the nucleic acid sample. In this method, the RG polynucleotide can be an RG1 polynucleotide, an RG2 polynucleotide, an RG3 polynucleotide, an RG4 polynucleotide, an RG5 polynucleotide or an RG7 polynucleotide. In this method, the RG resistance gene can be amplified prior to the step of contacting the nucleic acid sample with the RG polynucleotide, and, the RG resistance gene can be amplified by the polymerase chain reaction. In one embodiment, the RG polynucleotide is labeled.
The invention further provides for an RG polypeptide having at least 60% sequence identity to a polypeptide selected from the group consisting of: an RG1 polypeptide, an RG2 polypeptide, an RG3 polypeptide, an RG4 polypeptide, an RG5 polypeptide, and an RG7 polypeptide.
A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification, the figures and claims.
All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
This invention relates to families of RG genes, particularly from Lactuca sativa. Nucleic acid sequences of the present invention can be used to confer resistance in plants to a variety of pests including viruses, fungi, nematodes, insects, and bacteria. Sequences from within the RG genes can be used to fingerprint cultivars or germplasm for the presence of desired resistance genes. Promoters of RG genes can be used to drive heterologous gene expression under conditions in which RG genes are expressed. Further, the present invention provides RG proteins and antibodies specifically reactive to RG proteins. Antibodies to RG proteins can be used to detect the type and amount of RG protein expressed in a plant sample.
The present invention has use over a broad range of types of plants, including species from the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, and, Sorghum. In particularly preferred embodiments, species from the family Compositae and in particular the genus Lactuca are employed such as L. sativa and such subspecies as crispa, longifolia, and asparagina.
The nucleic acids of the present invention can be used in marker-aided selection. Marker-aided selection does not require the complete sequence of the gene or precise knowledge of which sequence confers which specificity. Instead, partial sequences can be used as hybridization probes or as the basis for oligonucleotide primers to amplify nucleic acid, e.g., by PCR. Partial sequences can be used in other methods, such as to follow the segregation of chromosome segments containing resistance genes in plants. Because the RG marker is the gene itself, there can be negligible recombination between the marker and the resistance phenotype. Thus, RG polynucleotides of the present invention provide an optimal means to DNA fingerprint cultivars and wild germplasm with respect to their disease resistance haplotypes. This can be used to indicate which germplasm accessions and cultivars carry the same resistance genes. At present, selection of plants (e.g., lettuce) for resistance to some diseases is slow and difficult. But linked markers allow indirect selection for such resistance genes. Moreover, RG markers also allow resistance genes to be identified and combined in a manner that would not otherwise be possible. Numerous accessions have been identified that provide resistance to all isolates of downy mildew (Bremia lactucae). However, without molecular markers it is impossible to combine such resistances from different sources. The nucleic acid sequences of the invention provide for a fast and convenient means to identify and combine resistances from different sources. The RG markers of the invention can also be used to identify recombinants that have new combinations of resistance genes in cis on the same chromosome.
In addition, RG markers may allow the identification of the Mendelian factors determining traits, such as field resistance to downy mildew. Once such markers have been identified, they will greatly increase the ease with which field resistance can be transferred between lines and combined with other resistances.
In another application, primers to RG sequences can be also designed to amplify sequences that are conserved in multiple RG family members. This gives genetic information on multiple RG family members. Alternatively, one or more primers can be made to sequences unique to a single resistance gene genus or a single RG specie. This allows an analysis of individual family groups (an RG genus) or an individual family member (a specie). Primers made to individual RGs at the edge of each cluster can be used to select for recombinants within the cluster. This minimizes the amount of linkage drag during introgression. Classical and molecular genetics has shown that pest resistance genes tend to be clustered in the genome. Pest resistance loci comprise arrays of genes and exhibit a variety of complex haplotypes rather than being simple alternate allelic forms. Pest resistance is conferred by families, or genuses, of related RG sequences, individual members, or species, of which have evolved to have a different specificity. Oligonucleotide primers can be designed that amplify members from multiple haplotypes, or genuses, or amplify only members of one genus, or only amplify an individual specie. This will provide codominant information and allow heterozygotes to be distinguished from homozygotes.
Further, comparison of RG sequences will allow a determination of which sequences are critical for resistance and will ultimately lead to engineering resistance genes with new specificities. Resistance gene sequences were not previously available for lettuce. Marker-aided selection will greatly increase the precision and speed of breeding for disease resistance. Transgenic approaches will allow pyramiding of resistance genes into a single Mendelian unit, transfer between sexually-incompatible species, substitute for conventional backcrossing procedures, and allow expression of other genes in parallel with resistance genes.
The RG polynucleotides also have utility in the construction of disease resistant transgenic plants. This avoids lengthy and sometimes difficult backcrossing programs currently necessary for introgression of resistance. It is also possible to transfer resistance polynucleotides between sexually-incompatible species, thereby greatly increasing the germplasm pool that can be used as a source of resistance genes. Cloning of multiple RG sequences in a single cassette will allow pyramiding of genes for resistance against multiple isolates of a single pathogen such as downy mildew or against multiple pathogens. Once introduced, such a cassette can be manipulated by classical breeding methods as a single Mendelian unit.
Transgenic plants of the present invention can also be constructed using an RG promoter. The promoter sequences from RG sequences of the invention can be used with RG genes or heterologous genes. Thus, RG promoters can be used to express a variety of genes in the same temporal and spatial patterns and at similar levels to resistance genes.
RG Polynucleotide Families
The present invention provides isolated nucleic acid constructs which comprise an RG polynucleotide. In alternative embodiments, the RG polynucleotide is at least 18 nucleotides in length, typically at least 20, 25, or 30 nucleotides in length, more typically at least 100 nucleotides in length, generally at least 200 nucleotides in length, preferably at least 300 nucleotides in length, more preferably at least 400 nucleotides in length, and most preferably at least 500 nucleotides in length.
In particularly preferred embodiments, the RG polynucleotide encodes a RG protein which confers resistance to plant pests. This RG protein can be longer, equivalent, or shorter than the RG protein encoded by an RG gene. In various embodiments, an RG polynucleotide can hybridize under stringent conditions to members of an RG family (an RG genus); e.g., it can hybridize to a member of the RG1 RG family, such as an RG1 polynucleotide selected from the group consisting of: SEQ ID NO:1 (RG1A); SEQ ID NO:2 and SEQ ID NO:137 (RG1B); SEQ ID NO:3 (RG1C); SEQ ID NO:4 (RG1D); SEQ ID NO:5 (RG1E); SEQ ID NO:6 (RG1F); SEQ ID NO:7 (RG1G); SEQ ID NO:8 (RG1H); SEQ ID NO:9 (RG1I) and SEQ ID NO:10 (RG1J).
In other embodiments, the polynucleotide can also hybridize under stringent conditions to a member of the RG2 family; such as an RG2 polynucleotide selected from the group consisting of: SEQ ID NO:21 and SEQ ID NO:27 (RG2A); SEQ ID NO:23 and SEQ ID NO:28 (RG2B); SEQ ID NO:29 (RG2C); SEQ ID NO:30 (RG2D); SEQ ID NO:31 (RG2E); SEQ ID NO:32 (RG2F); SEQ ID NO:33 (RG2G); SEQ ID NO:34 (RG2H); SEQ ID NO:35 (RG2I); SEQ ID NO:36 (RG2J); SEQ ID NO:37 (RG2K); SEQ ID NO:38 (RG2L); SEQ ID NO:39 (RG2M); SEQ ID NO:87 (RG2A); SEQ ID NO:89 (RG2B); SEQ ID NO:91 (RG2C); SEQ ID NO:93 (RG2D) and SEQ ID NO:94 (RG2D); SEQ ID NO:96 (RG2E); SEQ ID NO:98 (RG2F); SEQ ID NO:100 (RG2G); SEQ ID NO:102 (RG2H); SEQ ID NO:104 (RG2I); SEQ ID NO:106 (RG2J) and SEQ ID NO:107 (RG2J); SEQ ID NO:109 and SEQ ID NO:110(RG2K); SEQ ID NO:112 (RG2L); SEQ ID NO:114 (RG2M); SEQ ID NO:116 (RG2N); SEQ ID NO:118 (RG2O); SEQ ID NO:120 (RG2P); SEQ ID NO:122 (RG2Q); SEQ ID NO:124 (RG2S); SEQ ID NO:126 (RG2T); SEQ ID NO:128 (RG2U); SEQ ID NO:130 (RG2V); and, SEQ ID NO:132 (RG2W).
In alternative embodiments, each RG2 gene can also include an AC15 sequence which hybridizes under stringent conditions to a polynucleotide selected from the group consisting of: SEQ ID NO:56 (AC15-2A); SEQ ID NO:57 (AC15-2B); SEQ ID NO:58 (AC15-2C); SEQ ID NO:59 (AC15-2D); SEQ ID NO:60 (AC15-2E); SEQ ID NO:61 (AC15-2G); SEQ ID NO:62 (AC15-2H); SEQ ID NO:63 (AC15-21); SEQ ID NO:64 (AC15-2J); SEQ ID NO:65 (AC15-2L); SEQ ID NO:66 (AC15-2N); SEQ ID NO:67 (AC15-2O).
In other embodiments, an RG polynucleotide can hybridize under stringent conditions to an RG3 (SEQ ID NO:68), an RG4 (SEQ ID NO:69), and RG5 (SEQ ID NO:135), and an RG7 (SEQ ID NO:137), RG family member.
The present invention further provides nucleic acid constructs which comprise an RG polynucleotide which encodes RG polypeptides from various RG families; such as an RG polypeptide having at least 60% sequence identity to an RG polypeptide selected from the group consisting of: an RG1 polypeptide, an RG2 polypeptide, an RG3 polypeptide, and RG4 polypeptide, and RG5 polypeptide, and an RG7 polypeptide.
Exemplary RG1 polypeptides have the sequences shown in SEQ ID NO:2 (RG1A), SEQ ID NO:4 (RG1B), SEQ ID NO:6 (RG1C), SEQ ID NO:8 (RG1D), SEQ ID NO:10 (RG1E), SEQ ID NO:12 (RG1F), SEQ ID NO:14 (RG1G), SEQ ID NO:16 (RG1H), SEQ ID NO:20 (RG1J). Exemplary RG2 polypeptides have the sequences shown in SEQ ID NO:22 and SEQ ID NO:41 (RG2A); SEQ ID NO:24 and SEQ ID NO:42 (RG2B); SEQ ID NO:43 (RG2C); SEQ ID NO:44 (RG2D); SEQ ID NO:45 (RG2E); SEQ ID NO:46 (RG2F); SEQ ID NO:47 (RG2G); SEQ ID NO:48 (RG2H); SEQ ID NO:49 (RG2I); SEQ ID NO:50 (RG2J); SEQ ID NO:51 (RG2K); SEQ ID NO:52 (RG2L); SEQ ID NO:53 (RG2M); SEQ ID NO:88 (RG2A); SEQ ID NO:90 (RG2B); SEQ ID NO:92 (RG2C); SEQ ID NO:95 (RG2D); SEQ ID NO:97 (RG2E); SEQ ID NO:99 (RG2F); SEQ ID NO:101(RG2G); SEQ ID NO:103 (RG2H); SEQ ID NO:105 (RG2I); SEQ ID NO:108 (RG2J); SEQ ID NO:111 (RG2K); SEQ ID NO:113 (RG2L); SEQ ID NO:115 (RG2M); SEQ ID NO:117 (RG2N); SEQ ID NO:119 (RG2O); SEQ ID NO:121 (RG2P); SEQ ID NO:123 (RG2Q); SEQ ID NO:125 (RG2S); SEQ ID NO:127 (RG2T); SEQ ID NO:129 (RG2U); SEQ ID NO:131 (RG2V); and, SEQ ID NO:133 (RG2W).
An exemplary RG3 polypeptide has the sequence shown in SEQ ID NO:138. An exemplary RG4 polypeptide has the sequence shown in SEQ ID NO:139. RG polynucleotides will have at least 60% identity, more typically at least 65% identity, generally at least 70% identity, and preferably at least 75% identity, more preferably at least 80% identity, and most preferably at least 85%, 90%, or 95% identity at the deduced amino acid level. The regions where substantial identity is assessed can be inclusive or exclusive of the nucleotide binding site or the leucine rich region.
Vectors and Transcriptional Control Elements
The invention, providing methods and reagents for making novel species and genuses of RG nucleic acids described herein, further provides methods and reagents for expressing these nucleic acids using novel expression cassettes, vectors, transgenic plants and animals, using constitutive and inducible transcriptional and translational cis-(e.g., promoters and enhancers) and trans-acting control elements.
The expression of natural, recombinant or synthetic plant disease resistance polypeptide-encoding or other (i.e., antisense, ribozyme) nucleic acids can be achieved by operably linking the coding region a promoter (that can be plant-specific or not, constitutive or inducible), incorporating the construct into an expression cassette (such as an expression vector), and introducing the resultant construct into an in vitro reaction system or a suitable host cell or organism. Synthetic procedures may also be used. Typical expression systems contain, in addition to coding or antisense sequence, transcription and translation terminators, polyadenylation sequences, transcription and translation initiation sequences, and promoters useful for transcribing DNA into RNA. The expression systems optionally at least one independent terminator sequence, sequences permitting replication of the cassette in vivo, e.g., plants, eukaryotes, or prokaryotes, or a combination thereof, (e.g., shuttle vectors) and selection markers for the selected expression system, e.g., plant, prokaryotic or eukaryotic systems. To ensure proper polypeptide expression under varying conditions, a polyadenylation region at the 3xe2x80x2-end of the coding region can be included (see Li (1997) Plant Physiol. 115:321-325, for a review of the polyadenylation of RNA in plants). The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA (e.g., using Agrobacterium tumefaciens T-DNA replacement vectors, see e.g., Thykjaer (1997) Plant Mol Biol. 35:523-530; using a plasmid containing a gene of interest flanked by Agrobacterium T-DNA border repeat sequences; Hansen (1997) xe2x80x9cT-strand integration in maize protoplasts after codelivery of a T-DNA substrate and virulence genes,xe2x80x9d Proc. Natl. Acad. Sci. USA 94:11726-11730.
To identify the promoters, the 5xe2x80x2 portions of the clones described here are analyzed for sequences characteristic of promoter sequences. For instance, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site. In plants, further upstream from the TATA box, at positions xe2x88x9280 to xe2x88x92100, there is typically a promoter element with a series of adenines surrounding the trinucleotide G (or T) N G (see, e.g., Messing, in Genetic Engineering in Plants, pp. 221-227, Kosage, Meredith and Hollaender, eds. 1983). If proper polypeptide expression is desired, a polyadenylation region at the 3xe2x80x2-end of the RG coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from viral genes, such as T-DNA.
The nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses which are transiently expressed in cells using, for example, episomal expression systems (e.g., cauliflower mosaic virus (CAMV) viral RNA is generated in the nucleus by transcription of an episomal minichromosome containing supercoiled DNA, Covey (1990) Proc. Natl. Acad. Sci. USA 87:1633-1637). Alternatively, coding sequences can be inserted into the host cell genome becoming an integral part of the host chromosomal DNA.
Selection markers can be incorporated into expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences coding for episomal maintenance and replication such that integration into the host genome is not required. For example, the marker may encode biocide resistance, such as antibiotic resistance, particularly resistance to chloramphenicol, kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta, to permit selection of those cells transformed with the desired DNA sequences, see for example, Blondelet-Rouault (1997) Gene 190:315-317; Aubrecht (1997) J. Pharmacol. Exp. Ther. 281:992-997. Because selectable marker genes conferring resistance to substrates like neomycin or hygromycin can only be utilized in tissue culture, chemoresistance genes are also used as selectable markers in vitro and in vivo. See also, Mengiste (1997) xe2x80x9cHigh-efficiency transformation of Arabidopsis thaliana with a selectable marker gene regulated by the T-DNA 1xe2x80x2 promoter,xe2x80x9d Plant J. 12:945-948, showing that the 1xe2x80x2 promoter is an attractive alternative to the cauliflower mosaic virus (CaMV) 35S promoter for the generation of T-DNA insertion lines, the 1xe2x80x2 promoter may be especially beneficial for the secondary transformation of transgenic strains containing the 35S promoter to exclude homology-mediated gene silencing.
The endogenous promoters from the RG genes of the present invention can be used to direct expression of the genes. These promoters can also be used to direct expression of heterologous structural genes. The promoters can be used, for example, in recombinant expression cassettes to drive expression of genes conferring resistance to any number of pathogens or pests, including fungi, bacteria, and the like.
Constitutive Promoters
In construction of recombinant expression cassettes, vectors, transgenics, of the invention, a promoter fragment can be employed to direct expression of the desired gene in all tissues of a plant or animal. Promoters that drive expression continuously under physiological conditions are referred to as xe2x80x9cconstitutivexe2x80x9d promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include those from viruses which infect plants, such as the cauliflower mosaic virus (CaMV) 35S transcription initiation region; the 1xe2x80x2- or 2xe2x80x2-promoter derived from T-DNA of Agrobacterium tumafaciens; the promoter of the tobacco mosaic virus; and, other transcription initiation regions from various plant genes known to those of skill. See also Holtorf (1995) xe2x80x9cComparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana,xe2x80x9d Plant Mol. Biol. 29:637-646.
Inducible Promoters
Alternatively, a plant promoter may direct expression of the plant disease resistance nucleic acid of the invention under the influence of changing environmental conditions or developmental conditions. Examples of environmental conditions that may effect transcription by inducible promoters include pathogenic attack, anaerobic conditions, elevated temperature, drought, or the presence of light. Such promoters are referred to herein as xe2x80x9cinduciblexe2x80x9d promoters. For example, the invention incorporates the drought-inducible promoter of maize (Busk (1997) supra); the cold, drought, and high salt inducible promoter from potato (Kirch (1997) Plant Mol. Biol. 33:897-909).
Embodiments of the invention also incorporate use of plant promoters which are inducible upon injury or infection to express the invention""s plant disease resistance (RG) polypeptides. Various embodiments include use of, e.g., the promoter for a tobacco (Nicotiana tabacum) sesquiterpene cyclase gene (EAS4 promoter), which is expressed in wounded leafs, roots, and stem tissues, and upon infection with microbial pathogens (Yin (1997) Plant Physiol. 115(2):437-451); the ORF13 promoter from Agrobacteinum rhizogenes 8196, which is wound inducible in a limited area adjacent to the wound site (Hansen (1997) Mol. Gen. Genet. 254:337-343); the Shpx6b gene promoter, which is a plant peroxidase gene promoter induced by microbial pathogens (demonstrated using a fungal pathogen, see Curtis (1997) Mol. Plant Microbe Interact. 10:326-338); the wound-inducible gene promoter wun1, derived from potato (Siebertz (1989) Plant Cell 1:961-968); the wound-inducible Agrobacterium pmas gene (mannopine synthesis gene) promoter (Guevara-Garcia (1993) Plant J. 4:495-505).
Alternatively, plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the nucleic acids of the invention. For example, the invention can use the auxin-response elements E1 promoter fragment (AuxREs) in the soybean (Glycine mar L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) Mol. Plant Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274:1900-1902).
Plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics, are also used to express the nucleic acids of the invention. For example, the maize In2xe2x80x942 promoter, activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. Coding sequence can be under the control of, e.g., a tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11:1315-1324. Using chemically- (e.g., hormone- or pesticide-) induced promoters, harvesting of fruits and plant parts would be greatly facilitated. A chemical which can be applied to the transgenic plant in the field and induce expression of a polypeptide of the invention throughout all or most of the plant would make a environmentally safe defoliant or herbicide. Thus, the invention also provides for transgenic plants containing an inducible gene encoding for the RG polypeptides of the invention whose host range is limited to target plant species, such as weeds or crops before, during or after harvesting.
Abcission promoters are activated upon plant ripening, such as fruit ripening, and are especially useful incorporated in the expression systems (e.g., expression cassettes, vectors) of the invention. In some embodiments, when a plant disease resistant polypeptide-encoding nucleic acid is under the control of such a promoter, rapid cell death, induced by expression of the invention""s polypeptide, can accelerate and/or accentuate abcission, increasing the efficiency of the harvesting of fruits or other plant parts, such as cotton, and the like. Induction of rapid cell death at this time would accelerate separation of the fruit from the plant, greatly augmenting harvesting procedures. See, e.g., Kalaitzis (1997) Plant Physiol. 113:1303-1308, discussing tomato leaf and flower abscission; Payton (1996) Plant Mol. Biol. 31:1227-1231, discussing ethylene receptor expression regulation during fruit ripening, flower senescence and abscission; Koehler (1996) Plant Mol. Biol. 31:595-606, discussing the gene promoter for a bean abscission cellulase; Kalaitzis (1995) Plant Mol. Biol.28: 647-656, discussing cloning of a tomato polygalacturonase expressed in abscission; del Campillo (1996) Plant Physiol. 111:813-820, discussing pedicel breakstrength and cellulase gene expression during tomato flower abscission.
Tissue-Specific Promoters
Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues. Plant promoters which are active only in specific tissues or at specific times during plant development are used to express the nucleic acids of the invention. Examples of promoters under developmental control include promoters that initiate transcription only in certain tissues, such as leaves, roots, fruit, seeds, ovules, pollen, pistols, or flowers. Such promoters are referred to as xe2x80x9ctissue specificxe2x80x9d. The operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.
For example, a seed-specific promoter directs expression in seed tissues. Such promoters may be, for example, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed coat-specific, or some combination thereof. A leaf-specific promoter has been identified in maize, Busk (1997) Plant J. 11: 1285-1295. The ORF13 promoter from Agrobacterium rhizogenes exhibits high activity in roots (Hansen (1997) supra). A maize pollen-specific promoter has been identified in maize (Guerrero (1990) Mol. Gen. Genet. 224:161-168). A tomato promoter active during fruit ripening, senescence and abscission of leaves and, to a lesser extent, of flowers can be used (Blume (1997) Plant J. 12:731-746). A pistol specific promoter has been identified in the potato (Solanum tuberosum L.) SK2 gene, encoding a pistil-specific basic endochitinase (Ficker (1997) Plant Mol. Biol. 35:425-431). The Blec4 gene from pea (Pisum sativum cv. Alaska) is active in epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa, making it a useful tool to target the expression of foreign genes to the epidermal layer of actively growing shoots. The activity of the Blec4 promoter in the epidermis of the shoot apex makes it particularly suitable for genetically engineering defense against insects and diseases that attack the growing shoot apex (Mandaci (1997) Plant Mol Biol. 34:961-965).
The invention also provides for use of tissue-specific plant promoters include a promoter from the ovule-specific BELI gene described in Reiser (1995) Cell 83:735-742, GenBank No. U39944. Suitable seed specific promoters are derived from the following genes: MAC1 from maize, Sheridan (1996) Genetics 142:1009-1020; Cat3 from maize, GenBank No. L05934, Abler (1993)
Plant Mol. Biol. 22:10131-1038; the gene encoding oleosin 18 kD from maize, GenBank No. J05212, Lee (1994) Plant Mol. Biol. 26:1981-1987; vivparous-1 from Arabidopsis, Genbank No. U93215; the gene encoding oleosin from Arabidopsis, Genbank No. Z17657; Atmyc1 from Arabidopsis, Urao (1996) Plant Mol. Biol. 32:571-576; the 2s seed storage protein gene family from Arabidopsis, Conceicao (1994) Plant 5:493-505; the gene encoding oleosin 20 kD from Brassica napus, GenBank No. M63985; napA from Brassica napus, GenBank No. J02798, Josefsson (1987) JBL 26:12196-1301; the napin gene family from Brassica napus, Sjodahl (1995) Planta 197:264-271; the gene encoding the 2S storage protein from Brassica napus, Dasgupta (1993) Gene 133:301-302; the genes encoding oleosin a, Genbank No. U09118, and, oleosin B, Genbank No. U09119, from soybean; and, the gene encoding low molecular weight sulphur rich protein from soybean, Choi (1995) Mol Gen, Genet. 246:266-268. The tissue specific E8 promoter from tomato is particularly useful for directing gene expression so that a desired gene product is located in fruits. Other suitable promoters include those from genes encoding embryonic storage proteins.
One of skill will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well.
The invention also provides for use of tissue-specific promoters derived from viruses which can include, e.g., the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; the rice tungro bacilliform virus (RTBV), which replicates only in phloem cells in infected rice plants, with its promoter which drives strong phloem-specific reporter gene expression; the cassava vein mosaic virus (CVMV) promoter, with highest activity in vascular elements, in leaf mesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol. 31:1129-1139).
In some embodiments, the nucleic acid construct will comprise a promoter functional in a specific plant cell, such as in a species of Lactuca, operably linked to an RG polynucleotide. Promoters useful in these embodiments include RG promoters. In additional embodiments, the nucleic acid construct will comprise a RG promoter operably linked to a heterologous polynucleotide. The heterologous polynucleotide is chosen to provide a plant with a desired phenotype. For example, the heterologous polynucleotide can be a structural gene which encodes a polypeptide which imparts a desired resistance phenotype. Alternatively, the heterologous polynucleotide may be a regulatory gene which might play a role in transcriptional and/or translational control to suppress, enhance, or otherwise modify the transcription and/or expression of an endogenous gene within the plant. The heterologous polynucleotide of the nucleic acid construct of the present invention can be expressed in either sense or anti-sense orientation as desired. It will be appreciated that control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable plant characteristics.
Modifying and Inhibiting RG Gene Expression
The invention also provides for RG nucleic acid sequences which are complementary to the RG polypeptide-encoding sequences of the invention; i.e., antisense RG nucleic acids. Antisense technology can be conveniently used to modify gene expression in plants. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed. The construct is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been shown that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy (1988) Proc. Nat. Acad. Sci. USA 85:8805-8809; Hiatt et al., U.S. Pat. No. 4,801,340.
Antisense sequences are capable of inhibiting the transport, splicing or transcription of RG-encoding genes. The inhibition can be effected through the targeting of genomic DNA or messenger RNA. The transcription or function of targeted nucleic acid can be inhibited, e.g., by hybridization and/or cleavage. One particularly useful set of inhibitors provided by the present invention includes oligonucleotides which are able to either bind RG gene or message, in either case preventing or inhibiting the production or function of RG. The association can be though sequence specific hybridization. Such inhibitory nucleic acid sequences can, for example, be used to completely inhibit a plant disease resistance response. Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of RG message. The oligonucleotide can have enzyme activity which causes such cleavage, such as ribozymes. The oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid. One may screen a pool of many different such oligonucleotides for those with the desired activity.
Antisense Oligonucleotides
The invention provides for with antisense oligonucleotides capable of binding RG message which can inhibit RG activity by targeting mRNA. Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such RG oligonucleotides using the novel reagents of the invention. In some situations, naturally occurring nucleic acids used as antisense oligonucleotides may need to be relatively long (18 to 40 nucleotides) and present at high concentrations. A wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996). Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3xe2x80x2-thioacetal, methylene(methylimino), 3xe2x80x2-N-carbamate, and morpholino carbamate nucleic acids, as described herein.
Combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the sense and antisense RG sequences of the invention (for general background information, see, e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).
Inhibitory Ribozymes
The invention provides for with ribozymes capable of binding RG message which can inhibit RG activity by targeting mRNA. Strategies for designing ribozymes and selecting the RG-specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such RG ribozymes using the novel reagents of the invention. Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence, or, preventing transport of the message from the nucleus to the cytoplasm. After a ribozyme has bound and cleaved its RNA target, it is typically released from that RNA and so can bind and cleave new targets repeatedly.
Catalytic RNA molecules or ribozymes can also be used to inhibit expression of any plant gene. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described, e.g., in Haseloff (1988) Nature 334:585-591.
In some circumstances, the enzymatic nature of a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide. This potential advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.
The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RNaseP-like RNA (in association with an RNA guide sequence). Examples of such hammerhead motifs are described by Rossi (1992) Aids Research and Human Retroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S. Pat. No. 4,987,071. The recitation of these specific motifs is not intended to be limiting; those skilled in the art will recognize that an enzymatic RNA molecule of this invention has a specific substrate binding site complementary to one or more of the target gene RNA regions, and has nucleotide sequence within or surrounding that substrate binding site which imparts an RNA cleaving activity to the molecule.
Sense Supression
Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990), and U.S. Pat. No. 5,034,323.
Cloning of RG Polypeptides
Synthesis and/or cloning of RG polynucleotides and isolated nucleic acid constructs of the present invention are provided by methods well known to those of ordinary skill in the art. Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer""s specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989).
The isolation of RG genes may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. To prepare a cDNA library, mRNA is isolated from the desired organ, such as roots and a cDNA library which contains the RG gene transcript is prepared from the mRNA. Alternatively, cDNA may be prepared from mRNA extracted from other tissues in which RG genes or homologs are expressed.
The cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned RG gene such as the genes disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species.
Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through manipulation of the concentration of formamide within the range of 0% to 50%.
Alternatively, the RG nucleic acids of the invention can be amplified from nucleic acid samples using a variety of amplification techniques, such as polymerase chain reaction (PCR) technology, to amplify the sequences of the RG and related genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
Oligonucleotides can be used to identify and detect additional RG families and RG family species using a variety of hybridization techniques and conditions. Suitable amplification methods include, but are not limited to: polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y. (Innis )), ligase chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173); and, self-sustained sequence replication (Guatelli (1990) Proc. Natl. Acad. Sci. USA, 87:1874); Q Beta replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see Berger (1987) Methods Enzymol. 152:307-316, Sambrook, and Ausubel, as well as Mullis (1987) U.S. Pat. Nos. 4,683,195 and 4,683,202; Arnheim (1990) CandEN 36-47; Lomell J. Clin. Chem., 35:1826 (1989); Van Brunt, Biotechnology, 8:291-294 (1990); Wu (1989) Gene 4:560; Sooknanan (1995) Biotechnology 13:563-564. Methods for cloning in vitro amplified nucleic acids are described in Wallace, U.S. Pat. No. 5,426,039.
The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100 percent; however, it should be understood that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium as described earlier.
In some preferred embodiments, members of this class of pest resistance genes can be identified by their ability to be amplified by PCR primers based on the sequences disclosed here. Appropriate primers and probes for identifying RG sequences from plant tissues are generated from comparisons of the sequences provided herein. See, e.g., Table 1. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990), incorporated herein by reference.
Briefly, the first step of each cycle of the PCR involves the separation of the nucleic acid duplex formed by the primer extension. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complementary copies of the target strands. For successful PCR amplification, the primers are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer. The cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.
In the preferred embodiment of the PCR process, strand separation is achieved by heating the reaction to a sufficiently high temperature for an sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase (see U.S. Pat. No. 4,965,188). Template-dependent extension of primers in PCR is catalyzed by a polymerizing agent in the presence of adequate amounts of four deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and dTTP) in a reaction medium comprised of the appropriate salts, metal cations, and pH buffering system. Suitable polymerizing agents are enzymes known to catalyze template-dependent DNA synthesis.
Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
RG Proteins
The present invention further provides isolated RG proteins encoded by the RG polynucleotides disclosed herein. One of skill will recognize that the nucleic acid encoding a functional RG protein need not have a sequence identical to the exemplified genes disclosed here. For example, because of codon degeneracy a large number of nucleic acid sequences can encode the same polypeptide. In addition, the polypeptides encoded by the RG genes, like other proteins, have different domains which perform different functions. Thus, the RG gene sequences need not be full length, so long as the desired functional domain of the protein is expressed.
The resistance proteins are at least 25 amino acid residues in length. Typically, the RG proteins are at least 50 amino acid residues, generally at least 100, preferably at least 150, more preferably at least 200 amino acids in length. In particularly preferred embodiments, the RG proteins are of sufficient length to provide resistance to pests when expressed in the desired plants. Generally then, the RG proteins will be the length encoded by an RG gene of the present invention. However, those of ordinary skill will appreciate that minor deletions, substitutions, or additions to an RG protein will typically yield a protein with pest resistance characteristics similar or identical to that of the full length sequence. Thus, full-length RG proteins modified by 1, 2, 3, 4, or 5 deletions, substitutions, or additions, generally provide an effective degree of pest resistance relative to the full-length protein.
The RG proteins which provide pest resistance will typically comprise at least one of an LRR or an NBS. Preferably, both are present. LRR and/or NBS regions present in the RG proteins of the present invention can be provided by RG genes of the present invention. In some embodiments, the LRR and/or NBS regions are obtained from other pest resistance genes. See, e.g., Yu et al., Proc. Natl. Acad. Sci. USA, 93: 11751-11756 (1996); Bent et al., Science, 265: 1856-1860 (1994).
Modified protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art. For example, the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. Modification can also include swapping domains from the proteins of the invention with related domains from other pest resistance genes.
Pests that can be targeted by RG genes and proteins of the present invention include such bacterial pests as Erwinia carotovora and Pseudomonas marginalis. Fungal pests which can be targeted by the present invention include Bremia lactucae, Marssonina panattoniana, Rhizoctonia solani, Olpidium brassicae, root aphid, Sclerotinia sclerotiorum and S. minor, and Botrytis cinerea which causes gray mold. RG genes also provide resistance to viral diseases such as lettuce and turnip mosaic viruses.
Fusion Proteins
RG polypeptides can also be expressed as recombinant proteins with one or more additional polypeptide domains linked thereto to facilitate protein detection, purification, or other applications. Such detection and purification facilitating domains include, but are not limited to, metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein a domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). The inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between the purification domain and plant disease resistant polypeptide may be useful to facilitate purification. One such expression vector provides for expression of a fusion protein comprising the sequence encoding a plant disease resistant polypeptide of the invention and nucleic acid sequence encoding six histidine residues followed by thioredoxin and an enterokinase cleavage site (e.g., see Williams (1995) Biochemistry 34:1787-1797). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the desired protein(s) from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
Antibodies Reactive to RG Polypeptides and Immunological Assays
The present invention also provides antibodies which specifically react with RG proteins of the present invention under immunologically reactive conditions. An antibody immunologically reactive with a particular antigen can be generated in vivo or by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors. xe2x80x9cImmunologically reactive conditionsxe2x80x9d includes reference to conditions which allow an antibody, generated to a particular epitope of an antigen, to bind to that epitope to a detectably greater degree than the antibody binds to substantially all other epitopes, generally at least two times above background binding, preferably at least five times above background. Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols.
xe2x80x9cAntibodyxe2x80x9d includes reference to an immunoglobulin molecule obtained by in vitro or in vivo generation of the humoral response, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), and recombinant single chain Fv fragments (scFv). The term xe2x80x9cantibodyxe2x80x9d also includes antigen binding forms of antibodies (e.g., Fabxe2x80x2, F(abxe2x80x2)2, Fab, Fv, rIgG, and, inverted IgG). See, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.). An antibody immunologically reactive with a particular antigen can be generated in vivo or by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors. See, e.g., Huse et al. (1989) Science 246:1275-1281; and Ward, et al. (1989) Nature 341:544-546; and Vaughan et al. (1996) Nature Biotechnology, 14:309-314.
Many methods of making antibodies are known to persons of skill. A number of immunogens are used to produce antibodies specifically reactive to an isolated RG protein of the present invention under immunologically reactive conditions. An isolated recombinant, synthetic, or native RG protein of the present invention is the preferred immunogens (antigen) for the production of monoclonal or polyclonal antibodies.
The RG protein is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the RG protein. Methods of producing monoclonal or polyclonal antibodies are known to those of skill in the art. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, N.Y.); Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y.
Frequently, the RG proteins and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
The antibodies of the present invention can be used to screen plants for the expression of RG proteins of the present invention. The antibodies of this invention are also used for affinity chromatography in isolating RG protein.
The present invention further provides RG polypeptides that specifically bind, under immunologically reactive conditions, to an antibody generated against a defined immunogen, such as an immunogen consisting of the RG polypeptides of the present invention. Immunogens will generally be at least 10 contiguous amino acids from an RG polypeptide of the present invention. Optionally, immunogens can be from regions exclusive of the NBS and/or LRR regions of the RG polypeptides. Nucleic acids which encode such cross-reactive RG polypeptides are also provided by the present invention. The RG polypeptides can be isolated from any number plants as discussed earlier. Preferred are species from the family Compositae and in particular the genus Lactuca such as L. sativa and such subspecies as crispa, longifolia, and asparagina.
xe2x80x9cSpecifically bindsxe2x80x9d includes reference to the preferential association of a ligand, in whole or part, with a particular target molecule (i.e., xe2x80x9cbinding partnerxe2x80x9d or xe2x80x9cbinding moietyxe2x80x9d) relative to compositions lacking that target molecule. It is, of course, recognized that a certain degree of non-specific interaction may occur between a ligand and a non-target molecule. Nevertheless, specific binding, may be distinguished as mediated through specific recognition of the target molecule. Typically specific binding results in a much stronger association between the ligand and the target molecule than between the ligand and non-target molecule. Specific binding by an antibody to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. The affinity constant of the antibody binding site for its cognate monovalent antigen is at least 107, usually at least 108, preferably at least 109, more preferably at least 1010, and most preferably at least 1011 liters/mole. A variety of immunoassay formats are appropriate for selecting antibodies specifically reactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically reactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific reactivity. The antibody may be polyclonal but preferably is monoclonal. Generally, antibodies cross-reactive to such proteins as RPS2, RPM1 (bacterial resistances in Arabidopsis, L6 (fungal resistance in flax, PRF (resistance to Pseudomonas syringae in tomator), and N, (virus resistance in tobacco), are removed by immunoabsorbtion.
Immunoassays in the competitive binding format are typically used for cross-reactivity determinations. For example, an immunogenic RG polypeptide is immobilized to a solid support. Polypeptides added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above polypeptides to compete with the binding of the antisera to the immobilized RG polypeptide is compared to the immunogenic RG polypeptide. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with such proteins as RPS2, RPM1, L6, PRF, and N, are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorbtion with these non-RG resistance proteins.
The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay to compare a second xe2x80x9ctargetxe2x80x9d polypeptide to the immunogenic polypeptide. In order to make this comparison, the two polypeptides are each assayed at a wide range of concentrations and the amount of each polypeptide required to inhibit 50% of the binding of the antisera to the immobilized protein is determined using standard techniques. If the amount of the target polypeptide required is less than twice the amount of the immunogenic polypeptide that is required, then the target polypeptide is said to specifically bind to an antibody generated to the immunogenic protein. As a final determination of specificity, the pooled antisera is fully immunosorbed with the immunogenic polypeptide until no binding to the polypeptide used in the immunosorbtion is detectable. The fully immunosorbed antisera is then tested for reactivity with the test polypeptide. If no reactivity is observed, then the test polypeptide is specifically bound by the antisera elicited by the immunogenic protein.
Production Of Transgenic Plants Of The Invention
Isolated nucleic acid constructs prepared as described herein can be introduced into plants according techniques known in the art. In some embodiments, the introduced nucleic acid is used to provide RG gene expression and therefore pest resistance in desired plants. In some embodiments, RG promoters are used to drive expression of desired heterologous genes in plants. Finally, in some embodiments, the constructs can be used to suppress expression of a target endogenous gene, including RG genes.
To use isolated RG sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, for example, Weising et al. Ann. Rev. Genet. 22:421-477 (1988).
A DNA sequence coding for the desired RG polypeptide, for example a cDNA or a genomic sequence encoding a full length protein, will be used to construct a recombinant expression cassette which can be introduced into the desired plant. An expression cassette will typically comprise the RG polynucleotide operably linked to transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the RG gene in the intended tissues of the transformed plant.
Such DNA constructs may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, PEG poration, particle bombardment and microinjection of plant cell protoplasts or embryogenic callus, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.
Transformation techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. Embo J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987).
Agrobacterium tumefaciens-meditated transformation techniques are well described in the scientific literature. See, for example Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983). Although Agrobacterium is useful primarily in dicots, certain monocots can be transformed by Agrobacterium. For instance, Agrobacterium transformation of rice is described by Hiei et al, Plant J. 6:271-282 (1994). A particularly preferred means of transforming lettuce is described in Michelmore et al., Plant Cell Reports, 6:439-442 (1987).
Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired RG-controlled phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the RG nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, Macmillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).
The methods of the present invention are particularly useful for incorporating the RG polynucleotides into transformed plants in ways and under circumstances which are not found naturally. In particular, the RG polypeptides may be expressed at times or in quantities which are not characteristic of natural plants.
One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
Detection of RG Resistance Genes
The present invention further provides methods for detecting RG resistance genes in a nucleic acid sample suspected of comprising an RG resistance gene. The means by which the RG resistance gene is detected is not a critical aspect of the invention. For example, RG resistance genes can be detected by the presence of amplicons using RG resistance gene specific primers. Additionally, RG resistance genes can be detected by assaying for specific hybridization of an RG polynucleotide to an RG resistance gene. In some embodiments, the RG resistance gene can be amplified prior to the step of contacting the nucleic acid sample with the RG polynucleotide.
In a typical detection method, the nucleic acid sample is contacted with an RG polynucleotide to form a hybridization complex. The hybridization complex may be detected directly (e.g., in Southern or northern blots), or indirectly (e.g., by subsequent primer extension during PCR amplification). The RG polynucleotide hybridizes under stringent conditions to an RG polynucleotide of the invention. Formation of the hybridization complex is directly or indirectly used to indicate the presence of the RG resistance gene in the nucleic acid sample.
Detection of the hybridization complex can be achieved using any number of well known methods. For example, the nucleic acid sample, or a portion thereof, may be assayed by hybridization formats including but not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays. Briefly, in solution (or liquid) phase hybridizations, both the target nucleic acid and the probe or primer are free to interact in the reaction mixture. In solid phase hybridization assays, probes or primers are typically linked to a solid support where they are available for hybridization with target nucleic in solution. In mixed phase, nucleic acid intermediates in solution hybridize to target nucleic acids in solution as well as to a nucleic acid linked to a solid support. In in situ hybridization, the target nucleic acid is liberated from its cellular surroundings in such as to be available for hybridization within the cell while preserving the cellular morphology for subsequent interpretation and analysis. The following articles provide an overview of the various hybridization assay formats: Singer et al., Biotechniques 4(3):230-250 (1986); Haase et al., Methods in Virology, Vol. VII, pp. 189-226 (1984); Wilkinson, xe2x80x9cThe theory and practice of in situ hybridizationxe2x80x9d In: In situ Hybridization, Ed. D. G. Wilkinson. IRL Press, Oxford University Press, Oxford; and Nucleic Acid Hybridization: A Practical Approach, Ed. Hames, B. D. and Higgins, S. J., IRL Press (1987).
The effect of the modification of RG gene expression can be measured by detection of increases or decreases in mRNA levels using, for instance, Northern blots. In addition, the phenotypic effects of gene expression can be detected by measuring nematode, fungal, bacterial, viral, or other pest resistance in plants. Suitable assays for determining pest resistance are well known. Michelmore and Crute, Trans. Br. mycol. Soc, 79(3):542-546 (1982).
The means by which hybridization complexes are detected is not a critical aspect of the present invention and can be accomplished by any number of methods currently known or later developed. RG polynucleotides can be labeled by any one of several methods typically used to detect the presence of hybridized nucleic acids. One common method of detection is the use of autoradiography using probes labeled with 3H, 125I, 35S, 14C, or 32P, or the like. The choice of radioactive isotope depends on research preferences due to ease of synthesis, stability, and half lives of the selected isotopes. Other labels include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. Alternatively, probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents or enzymes. The choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation. Labeling the RG polynucleotide is readily achieved such as by the use of labeled PCR primers.
The choice of label dictates the manner in which the label is bound to the probe. Radioactive probes are typically made using commercially available nucleotides containing the desired radioactive isotope. The radioactive nucleotides can be incorporated into probes, for example, by using DNA synthesizers, by nick translation with DNA polymerase I, by tailing radioactive DNA bases to the 3xe2x80x2 end of probes with terminal deoxynucleotidyl transferase, by treating single-stranded M13 plasmids having specific inserts with the Klenow fragment of DNA polymerase in the presence of radioactive deoxynucleotides, dNTP, by transcribing from RNA templates using reverse transcriptase in the presence of radioactive deoxynucleotides, dNTP, or by transcribing RNA from vectors containing specific RNA viral promoters (e.g., SP6 promoter) using the corresponding RNA polymerase (e.g., SP6 RNA polymerase) in the presence of radioactive ribonucleotides rNTP.
The probes can be labeled using radioactive nucleotides in which the isotope resides as a part of the nucleotide molecule, or in which the radioactive component is attached to the nucleotide via a terminal hydroxyl group that has been esterified to a radioactive component such as inorganic acids, e.g., 32P phosphate or 14C organic acids, or esterified to provide a linking group to the label. Base analogs having nucleophilic linking groups, such as primary amino groups, can also be linked to a label.
Non-radioactive probes are often labeled by indirect means. For example, a ligand molecule is covalently bound to the probe. The ligand then binds to an anti-ligand molecule which is either inherently detectable or covalently bound to a detectable signal system, such as an enzyme, a fluorophore, or a chemiluminescent compound. Enzymes of interest as labels will primarily be hydrolases, such as phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescers include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. Ligands and anti-ligands may be varied widely. Where a ligand has a natural anti-ligand, namely ligands such as biotin, thyroxine, and cortisol, it can be used in conjunction with its labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
Probes can also be labeled by direct conjugation with a label. For example, cloned DNA probes have been coupled directly to horseradish peroxidase or alkaline phosphatase, (Renz. M., and Kurz, K. (1984) A Colorimetric Method for DNA Hybridization. Nucl. Acids Res. 12: 3435-3444) and synthetic oligonucleotides have been coupled directly with alkaline phosphatase (Jablonski, E., et al. (1986) Preparation of Oligodeoxynucleotide-Alkaline Phosphatase Conjugates and Their Use as Hybridization Probes. Nuc. Acids. Res. 14: 6115-6128; and Li P., et al. (1987) Enzyme-linked Synthetic Oligonucleotide probes: Non-Radioactive Detection of Enterotoxigenic Escherichia Coli in Faeca Specimens. Nucl. Acids Res. 15:5275-5287).
Definitions
Units, prefixes, and symbols can be denoted in their SI accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5xe2x80x2 to 3xe2x80x2 orientation, respectively. The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.
As used herein, the term xe2x80x9cplantxe2x80x9d includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
As used herein, xe2x80x9cpestxe2x80x9d includes, but is not limited to, viruses, fungi, nematodes, insects, and bacteria.
As used herein, xe2x80x9cheterologousxe2x80x9d is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its original form. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form.
As used herein, xe2x80x9cRG gene,xe2x80x9d alternatively referred to as xe2x80x9cRLG gene,xe2x80x9d is a gene encoding resistance to plant pests, such as viruses, fungi, nematodes, insects, and bacteria, and which hybridizes under stringent conditions and/or has at least 60% sequence identity at the deduced amino acid level to the exemplified sequences provided herein. RG genes encode xe2x80x9cRG polypeptides,xe2x80x9d alternatively referred to as xe2x80x9cRLG polypeptides,xe2x80x9d which can comprise LRR motifs and/or NBS motifs. The RG polypeptides encoded by RG genes have at least 55% or 60% sequence identity, typically at least 65% sequence identity, preferably at least 70% sequence identity, often at least 75% sequence identity, more preferably at least 80% sequence identity, and most preferably at least 90% sequence identity at the deduced amino acid level relative to the exemplary RG sequences provided herein. The term xe2x80x9cRG familyxe2x80x9d or xe2x80x9cRG family genusxe2x80x9d or xe2x80x9cgenusxe2x80x9d includes reference to a group of RG polypeptide sequence species that have at least 60% amino acid sequence identity, and, the nucleic acids encoding these polypeptides. The individual species of a genus, i.e., the members of a family, typically are genetically mapped to the same locus.
As used herein, xe2x80x9cRG polynucleotidexe2x80x9d includes reference to a contiguous sequence from an RG gene of at least 18, 20, 25, 30, 40, or 50 nucleotides in length, up to at least about 100 or at least about 200 nucleotides in length. In some embodiments, the polynucleotide is preferably at least 100 nucleotides in length, more preferably at least 200 nucleotides in length, most preferably at least 500 nucleotides in length. Thus, RG polynucleotide may be a RG gene or a subsequence thereof.
As used herein, xe2x80x9cisolated,xe2x80x9d when referring to a molecule or composition, such as, for example, an RG polypeptide or nucleic acid, means that the molecule or composition is separated from at least one other compound, such as a protein, other nucleic acids (e.g., RNAS), or other contaminants with which it is associated in vivo or in its naturally occurring state. Thus, an RG polypeptide or nucleic acid is considered isolated when it has been isolated from any other component with which it is naturally associated, e.g., cell membrane, as in a cell extract. An isolated composition can, however, also be substantially pure. An isolated composition can be in a homogeneous state and can be in a dry or an aqueous solution. Purity and homogeneity can be determined, for example, using analytical chemistry techniques such as polyacrylamide gel electrophoresis (SDS-PAGE) or high performance liquid chromatography (HPLC).
The term xe2x80x9cnucleic acidxe2x80x9d or xe2x80x9cnucleic acid moleculexe2x80x9d or xe2x80x9cnucleic acid sequencexe2x80x9d refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved thereover for the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3xe2x80x2-thioacetal, methylene(methylimino), 3xe2x80x2-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197. Other synthetic backbones encompasses by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and amplification product. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof.
The term xe2x80x9cexogenous nucleic acidxe2x80x9d refers to a nucleic acid that has been isolated, synthesized, cloned, ligated, excised in conjunction with another nucleic acid, in a manner that is not found in nature, and/or introduced into and/or expressed in a cell or cellular environment other than or at levels or forms different than the cell or cellular environment in which said nucleic acid or protein is be found in nature. The term encompasses both nucleic acids originally obtained from a different organism or cell type than the cell type in which it is expressed, and also nucleic acids that are obtained from the same cell line as the cell line in which it is expressed invention.
The term xe2x80x9crecombinant,xe2x80x9d when used with reference to a cell, or to the nucleic acid, protein or vector refers to a material, or a material corresponding to the natural or native form of the material, that has been modified by the introduction of a new moiety or alteration of an existing moiety, or is identical thereto but produced or derived from synthetic materials. For example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level, typically, under-expressed or not expressed at all. The term xe2x80x9crecombinant meansxe2x80x9d encompasses all means of expressing, i.e., transcription or translation of, an isolated and/or cloned nucleic acid in vitro or in vivo. For example, the term xe2x80x9crecombinant meansxe2x80x9d encompasses techniques where a recombinant nucleic acid, such as a cDNA encoding a protein, is inserted into an expression vector, the vector is introduced into a cell and the cell expresses the protein. xe2x80x9cRecombinant meansxe2x80x9d also encompass the ligation of nucleic acids having coding or promoter sequences from different sources into one vector for expression of a fusion protein, constitutive expression of a protein, or inducible expression of a protein, such as the plant disease resistant, or RG, polypeptides of the invention.
The term xe2x80x9cspecifically hybridizesxe2x80x9d refers to a nucleic acid that hybridizes, duplexes or binds to a particular target DNA or RNA sequence. The target sequences can be present in a preparation of total cellular DNA or RNA. Proper annealing conditions depend, for example, upon a nucleic acid""s, such as a probe""s length, base composition, and the number of mismatches and their position on the probe, and can be readily determined empirically providing the appropriate reagents are available. For discussions of nucleic acid probe design and annealing conditions, see, e.g., Sambrook and Ausubel.
The terms xe2x80x9cstringent hybridization,xe2x80x9d xe2x80x9cstringent conditions,xe2x80x9d or xe2x80x9cspecific hybridization conditionsxe2x80x9d refers to conditions under which an oligonucleotide (when used, for example, as a probe or primer) will hybridize to its target subsequence, such as an RG nucleic acid in an expression vector of the invention but not to a non-RG sequence. Stringent conditions are sequence-dependent. Thus, in one set of stringent conditions an oligonucleotide probe will hybridize to only one specie of the genus of RG nucleic acids of the invention. In another set of stringent conditions (less stringent) an oligonucleotide probe will hybridize to all species of the invention""s genus but not to non-RG nucleic acids. Longer sequences hybridize specifically at higher temperatures. Stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium (if the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, i.e., about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Often, high stringency wash conditions preceded by low stringency wash conditions to remove background probe signal. An example of medium stringency wash conditions for a duplex of, e.g., more than 100 nucleotides, is 1xc3x97SSC at 45xc2x0 C. for 15 minutes (see Sambrook for a description of SSC buffer). An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4xc3x97SSC at 40xc2x0 C. for 15 minutes, a signal to noise ratio of 2xc3x97 (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a xe2x80x9cspecific hybridization.xe2x80x9d Nucleic acids which do not hybridize to each other under stringent conditions can still be substantially identical if the polypeptides which they encode are substantially identical. This can occurs, e.g., when a nucleic acid is created that encodes for conservative substitutions. Stringent hybridization and stringent hybridization wash conditions are different under different environmental parameters, such as for Southern and Northern hybridizations. An extensive guide to the hybridization of nucleic acids is found in, e.g., Sambrook, Tijssen (1993) supra.
As used herein xe2x80x9coperably linkedxe2x80x9d includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
In the expression of transgenes one of skill will recognize that the inserted polynucleotide sequence need not be identical and may be xe2x80x9csubstantially identicalxe2x80x9d to a sequence of the gene from which it was derived. As explained herein, these variants are specifically covered by this term.
In the case where the inserted polynucleotide sequence is transcribed and translated to produce a functional RG polypeptide, one of skill will recognize that because of codon degeneracy, a number of polynucleotide sequences will encode the same polypeptide. These variants are specifically covered by the term xe2x80x9cRG polynucleotide sequencexe2x80x9d. In addition, the term specifically includes those full length sequences substantially identical (determined as described herein) with an RG gene sequence which encode proteins that retain the function of the RG protein. Thus, in the case of RG genes disclosed here, the term includes variant polynucleotide sequences which have substantial identity with the sequences disclosed here and which encode proteins capable of conferring resistance to nematodes, bacteria, viruses, fungi, insects or other pests on a transgenic plant comprising the sequence.
Two polynucleotides or polypeptides are said to be xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence, as described below. The term xe2x80x9ccomplementary toxe2x80x9d is used herein to mean that the complementary sequence is identical to all or a specified contiguous portion of a reference polynucleotide sequence.
The terms xe2x80x9csequence identity,xe2x80x9d xe2x80x9csequence similarityxe2x80x9d and xe2x80x9chomologyxe2x80x9d refer to when two sequences, such as the nucleic acid and amino acid sequences or the polypeptides of the invention, when optimally aligned, as with, for example, the programs PILEUP, BLAST, GAP, FASTA or BESTFIT (see discussion, supra). xe2x80x9cPercentage amino acid/nucleic acid sequence identityxe2x80x9d refers to a comparison of the sequences of two polypeptides/nucleic acids which, when optimally aligned, have approximately the designated percentage of the same amino acids/nucleic acids, respectively. For example, xe2x80x9c60% sequence identityxe2x80x9d and xe2x80x9c60% homologyxe2x80x9d refer to a comparison of the sequences of two RG nucleic acids or polypeptides which, when optimally aligned, have 60% identity. For example, in one embodiment, nucleic acids encoding RG polypeptides of the invention comprise a sequence with at least 50% nucleic acid sequence identity to SEQ ID NO:1. In other embodiments, the RG polypeptides of the invention are encoded by nucleic acids comprising a sequence with at least 50% sequence identity to SEQ ID NO:1, or, are encoded by nucleic acids comprising SEQ ID NO:1, or, have at least 60% amino acid sequence identity to the polypeptide of SEQ ID NO:2.
xe2x80x9cPercentage of sequence identityxe2x80x9d is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The term xe2x80x9csubstantial identityxe2x80x9d of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 55% or 60% sequence identity, generally at least 65%, preferably at least 70%, often at least 75%, more preferably at least 80% and most preferably at least 90%, compared to a reference sequence using the programs described above (preferably BESTFIT) using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 55% or 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 95%. Polypeptides having xe2x80x9csequence similarityxe2x80x9d share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under appropriate conditions. Appropriate conditions can be high or low stringency and will be different in different circumstances. Generally, stringent conditions are selected to be about 5xc2x0 C. to about 20xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent wash conditions are those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 50xc2x0 C. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
Nucleic acids of the invention can be identified from a cDNA or genomic library prepared according to standard procedures and the nucleic acids disclosed here used as a probe. Thus, for example, stringent hybridization conditions will typically include at least one low stringency wash using 0.3 molar salt (e.g., 2xc3x97SSC) at 65xc2x0 C. The washes are preferably followed by one or more subsequent washes using 0.03 molar salt (e.g., 0.2xc3x97SSC) at 50xc2x0C., usually 60xc2x0 C., or more usually 65xc2x0 C. Nucleic acid probes used to identify the nucleic acids are preferably at least 100 nucleotides in length.
As used herein, xe2x80x9cnucleotide binding sitexe2x80x9d or xe2x80x9cnucleotide binding domainxe2x80x9d (xe2x80x9cNBSxe2x80x9d) includes reference to highly conserved nucleotide-, i.e., ATP/GTP-, binding domains, typically included in the xe2x80x9ckinase domainxe2x80x9d of kinase polypeptides, such as a kinase-la, kinase 2, or a kinase 3a motif, as described herein. For example, the tobacco N and Arabidopsis RPS2 genes, among several recently cloned disease-resistance genes, share highly conserved NBS sequence. Kinase NBS subdomains further consist of three subdomain motifs: the P-loop, kinase-2, and kinase-3a subdomains (Yu (1996) Proc. Acad. Sci. USA 93:11751-11756). As discussed in detail herein, examples include the Arabidopsis RPP5 gene (Parker (1997) supra), the A. thaliana RPS2 gene (Mindrinos (1997) supra), and the flax L6 rust resistance gene (Lawrence (1995) supra) which all encode proteins containing an NBS; and Mindrinos (1994) Cell 78:1089-1099; and Shen (1993) FEBS 335:380-385. Using the teachings disclosed and incorporated herein and standard nucleic acid hybridization and/or amplification techniques, one of skill can identify members having NBS domains, including any of the genus of NBS-containing plant disease resistant polypeptides of the invention.
As used herein, xe2x80x9cleucine rich regionxe2x80x9d (xe2x80x9cLRRxe2x80x9d) includes reference to a region that has a leucine content of at least 20% leucine or isoleucine, or 30% of the aliphatic residues: leucine, isoleucine, methionine, valine, and phenylalanine, and arranged with approximate repeated periodicity. The length of the repeat may vary in length but is generally about 20 to 30 amino acids. An LRR-containing polypeptide typically will have the canonical 24 amino acid leucine-rich repeat (LRR) sequence, which is present in different proteins that mediates molecular recognition and/or interaction processes; as described in Bent (1994) Science 265:1856-1860; Parker (1997) Plant Cell. 9:879-894; Hong (1997) Plant Physiol. 113:1203-1212; Schmitz (1997) Nucleic Acids Res. 25:756-763; Hipskind (1996) Mol. Plant Microbe Interact. 9:819-825; Tornero (1996) Plant J. 10:315-330; Dixon (1996) Cell 84:451-459; Jones (1994) Science 266:789-793; Lawrence (1995) Plant Cell 7:1195-1206; Song (1995) Science 270:1804-1806; as discussed in further detail supra. Using the teachings disclosed and incorporated herein and standard nucleic acid hybridization and/or amplification techniques, one of skill can identify polypeptides having LRR domains, including any member of the genus of LRR-containing RG polypeptides of the invention.
The term xe2x80x9cpromoterxe2x80x9d refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A xe2x80x9cplant promoterxe2x80x9d is a promoter capable of initiating and/or regulating transcription in plant cells; see also discussion on plant promoters, supra.
The term xe2x80x9cconstitutive promoterxe2x80x9d refers to a promoter that initiates and helps control transcription in all tissues. Promoters that drive expression continuously under physiological conditions are referred to herein as xe2x80x9cconstitutivexe2x80x9d promoters and are active under most environmental conditions and states of development or cell differentiation; see also detailed discussion, supra.
The term xe2x80x9cinducible promoterxe2x80x9d refers to a promoter which directs transcription under the influence of changing environmental conditions or developmental conditions. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light. Such promoters are referred to herein as xe2x80x9cinduciblexe2x80x9d promoters; see also detailed discussion, supra.
The term xe2x80x9cabscission-induced promoterxe2x80x9d or xe2x80x9cabcission promoterxe2x80x9d refers to a class of promoters which are activated upon plant ripening, such as fruit ripening, and are especially useful incorporated in the expression systems (e.g., expression cassettes, vectors) of the invention. When the plant disease resistant polypeptide-encoding nucleic acid is under the control of an abcission promoter, rapid cell death, induced by expression of the invention""s polypeptide, accelerates and/or accentuates abcission of the plant part, increasing the efficiency of the harvesting of fruits or other plant parts, such as cotton, and the like; see also detailed discussion, supra.
The term xe2x80x9ctissue-specific promoterxe2x80x9d refers to a class of transcriptional control elements that are only active in particular cells or tissues. Examples of plant promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as roots, leaves, fruit, ovules, seeds, pollen, pistols, or flowers; see also detailed discussion, supra.
As used herein xe2x80x9crecombinantxe2x80x9d includes reference to a cell, or nucleic acid, or vector, that has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid to a form not native to that cell, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
As used herein, a xe2x80x9crecombinant expression cassettexe2x80x9d or xe2x80x9cexpression cassettexe2x80x9d is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a target cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes a nucleic acid to be transcribed, and a promoter.
As used herein, xe2x80x9ctransgenic plantxe2x80x9d includes reference to a plant modified by introduction of a heterologous polynucleotide. Generally, the heterologous polynucleotide is an RG structural or regulatory gene or subsequences thereof.
As used herein, xe2x80x9chybridization complexxe2x80x9d includes reference to a duplex nucleic acid sequence formed by selective hybridization of two single-stranded nucleic acids with each other.
As used herein, xe2x80x9camplifiedxe2x80x9d includes reference to an increase in the molarity of a specified sequence. Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (SSR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well-known to persons of skill.
As used herein, xe2x80x9cnucleic acid samplexe2x80x9d includes reference to a specimen suspected of comprising RG resistance genes. Such specimens are generally derived, directly or indirectly, from lettuce tissue.
The term xe2x80x9cantibodyxe2x80x9d refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments or synthetic or recombinant analogues thereof which specifically bind and recognize analytes and antigens, such as a genus or subgenus of polypeptides of the invention, as described supra.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.